Cerebral vasospasm after subarachnoid hemorrhage (SAH) remains the greatest limitation to successful post-operative management of aneurysmal stroke. Despite intensive study, the basic mechanisms leading to "irreversible" chronic cerebroarterial constriction in response to subarachnoid (SA) blood clot remain unclear. This project seeks, as its long-term objective, intervention against a major cause of morbidity and mortality after aneurysm rupture. It has been long believed that erythrocytes in the SA clot are crucial to the development of cerebral vasospasm. Recent work in the canine model supports this idea and has led to a specific hypothesis for the mechanisms of human cerebral vasospasm. Testing of that hypothesis is the specific aim of this proposal. Central to the hypothesis is our finding that human erythrocytes incubated in vitro under conditions like those of the SA clot do not lyse rapidly, but become progressively denatured on a time-scale compatible with the onset of clinical pathology. Upon exposure to plasma complement proteins, such "aged" erythrocytes initiate the "alternate" pathway of complement activation, leading to formation of "membrane attack complex" and their own hemolysis. Additional actions of activated complement include stimulation of inflammatory infiltration and increased permeability of the blood-brain barrier. The proposed in vitro work will examine whether this finding is a general feature of human and canine erythrocytes. The canine model will be used to verify that denatured SA erythrocytes promote by complement protein activation two major components of cerebral vasospasm: inflammation and hemolysis. The role of local extravasation of plasma protein into the SA clot, which would then lead to rapid complement-induced hemolysis, will be examined by immunohistochemical and protein marker studies. The time- course of SA erythrocyte denaturation may well account for the clinical hallmark of cerebral vasospasm: delayed onset after SAH. The proposed studies represent a departure from previous efforts which emphasized vasoactive substances arising in the SA clot. By aiming at prevention of rapid SA erythrocyte lysis, a whole cycle of threatening events may be prevented.